Randomized Trial of Methylcobalamin and Folate Effects on Homocysteine in Hemodialysis Patients

Original Paper Nephron 2002;91:58 63 Accepted: July 10, 2001 Randomized Trial of Methylcobalamin and Folate Effects on Homocysteine in Hemodialysis Patients Hernán Trimarchi a Amalia Schiel b Emilio Freixas a Marisa Díaz b Departments of a Nephrology and b Nuclear Medicine, Hospital Británico de Buenos Aires, Argentina Key Words Chronic renal failure W Folate W Hemodialysis W Homocysteine W Hyperhomocysteinemia W Methylcobalamin Abstract Background: There are no data available on the effects of intravenous (i.v.) methylcobalamin (Me-Cbl), the coenzymatically active form of vitamin B 12 that acts as a cofactor for methionine synthase in the conversion of total homocysteine (thcy) to methionine, with or without oral folic acid (FA) supplementation, on fasting thcy levels in hemodialysis (HD) patients. Methods: We performed a prospective randomized trial in which 62 chronic HD patients without previous vitamin supplementation were divided into four groups. Group A received Me-Cbl 500 Ìg twice/week plus FA 10 mg/day; group B received FA 10 mg/day alone; group C received no vitamin supplementation, and group D was on Me-Cbl 500 Ìg twice/ week alone. Fasting thcy, vitamin B 12, serum (s) FA and erythrocytic (e) FA were measured predialysis before and after 4 months of therapy. Results: Final thcy levels were significantly lower in group A (10.2 B 3.1 Ìmol/l) compared to groups C (27.3 B 9.7 Ìmol/l, p! 0.001) and group D (24.3 B 11.8 Ìmol/l, p! 0.001) and similar to group B (11.2 B 1.9 Ìmol/l, p = n.s.). Mean thcy levels showed a significant decrease in group A from 22.5 B 15.6 to 10.2 B 3.1 Ìmol/l (p = 0.003) and in group B from 19.9 B 4.0 to 11.2 B 1.9 Ìmol/l (p = 0.012), while no significant changes were observed in groups C (25.9 B 9.3 vs. 27.3 B 9.7 Ìmol/l, p = n.s.) and D (26.6 B 14.3 vs. 24.3 B 11.8 Ìmol/l, p = n.s.). Conclusion: Oral FA (10 mg/day) supplementation appears to be an effective approach to normalize plasma thcy in chronic HD patients; the addition of i.v. Me-Cbl (500 Ìg twice/week) to this regimen showed no benefit. Separately, FA corrected hyperhomocysteinemia (HtHcy), while Me-Cbl showed no change. Introduction Copyright 2002 S. Karger AG, Basel Cardiovascular disease is the major cause of death in end-stage renal disease patients, due in part to a higher prevalence of established arteriosclerotic risk factors [1, 2]. Recently, controlled evidence has shown that HtHcy occurs more commonly than other cardiovascular risk factors in patients on chronic HD [3]. According to some reports, HtHcy, defined as fasting thcy levels 1 15 Ìmol/l [4, 5] and classified as moderate (15 30 Ìmol/l), intermediate (30 100 Ìmol/l), and severe (1100 Ìmol/l) [6], occurs in 83% of the dialysis population [7, 8], and appears to contribute independently to their excess incidence of cardiovascular outcomes [9 11]. Moreover, thcy disrupts ABC Fax + 41 61 306 12 34 E-Mail karger@karger.ch www.karger.com 2002 S. Karger AG, Basel 0028 2766/02/0911 0058$18.50/0 Accessible online at: www.karger.com/journals/nef Hernán Trimarchi, MD Hospital Británico de Buenos Aires Perdriel 74 1280 Buenos Aires (Argentina) Tel. +54 1 143096400, Fax +54 1 143043393, E-Mail htrimarchi@hotmail.com

Table 1. Characteristics of 62 hemodialysis patients divided into 4 groups Group Age years Males Time on HD months KTV Initial albumin, g/l Initial Hct % Group A (n = 17) Group B (n = 16) Group C (n = 13) Group D (n = 16) 59.70B18.1 (35 80) 62.2B10.6 (38 75) 50.7B18.6 (19 70) 58.1B21.2 (27 86) 10 28.3B21.4 (7 75) 10 13.6B9.2 (7 30) 7 20.9B15.2 (6 45) 8 12B9.7 (5 28) 1.2B0.3 3.6B0.2 32.3B3.6 1.2B0.3 3.8B0.2 32.3B3.2 1.2B0.2 3.7B0.1 32.6B2.8 1.2B0.3 3.4B0.5 29B6.9 HD = Hemodialysis; Hct = hematocrit. Note: All differences between groups are nonsignificant. several endothelium-related anticoagulant functions resulting in enhanced thrombogenicity and contributing to the occlusion of arterio-venous fistulas and grafts in HD patients [12 14]. More recently, a prospective study revealed that each 1 Ìmol/l increase in the thcy level was associated with a 4% increase in the risk of access thrombosis [15]. Folate, vitamin B 6 and vitamin B 12 are the main cofactors for thcy metabolism. Vitamin B 12 and folate play a critical role in the remethylation of homocysteine to methionine [16, 17]. In addition, in chronic renal failure patients a significant reduction in membrane protein methyl esterification exists, and structural cellular damages accumulate, rendering proteins inadequately repaired [18]. Me-Cbl, the methylated form of cobalamin, is the cofactor for the enzyme methionine synthase that converts homocysteine to methionine [19]. In the Me-Cbldependent [16] reaction mediated by methionine synthase, homocysteine acquires a methyl group from methyltetrahydrofolate to form methionine. A considerable proportion of methionine is then used to form S-adenosylmethionine, a universal donor of methyl groups [1, 20]. Therefore, it seems appropriate to diminish thcy levels particularly in HD patients. Several studies in HD patients have shown that FA supplementation at doses of 10 mg/day appear to lower fasting plasma thcy levels by 30 50% [21 23]. In our center, because many HD patients were not taking vitamins, we have randomly selected a group which was started on i.v. Me-Cbl to avoid noncompliance and putative absorption differences among patients and assess its direct effects on thcy alone or coupled with oral FA, overcoming the uremic-altered FAmediated methylation processes. Subjects and Methods We undertook a prospective, randomized trial in which predialysis fasting plasma concentrations of thcy, vitamin B 12, sfa and efa, were determined in 62 chronic HD patients before and after 4 months of therapy with i.v. Me-Cbl and/or FA supplementation or without treatment. In this prospective study, patients were randomly divided in a blinded fashion by a random-number generator, stratified only for centre, into four groups: group A (n = 17) received 500 Ìg of Me-Cbl (Methycobal ; Esai, Bunkyo-ku, Tokyo, Japan) postdialysis twice a week plus FA 10 mg/day; group B (n = 16) was on 10 mg of daily FA supplementation; group C (n = 13) received no vitamin therapy and group D (n = 16) received 500 Ìg of Me-Cbl twice weekly postdialysis. We included patients between 19 and 86 years of age, requiring HD for at least 5 months. Patients were excluded if previous vitamin supplementation was prescribed. All patients concluded the study. All patients received epoetin 4,000 U subcutaneously twice weekly and 100 mg of i.v. iron saccharate postdialysis when necessary to maintain transferrin saturation between 25 and 50%. All groups were not different according to age, to gender, to time on HD, to KT/V (table 1), to primary renal disease, to diet, and to baseline plasma levels of thcy, vitamin B 12, sfa, efa, albumin and hematocrits (tables 2, 3). Thrice weekly high-flux HD was performed using polysulphone (F80, Fresenius AG, Bad Homburg, Germany) or triacetate cellulose (FB 210 U, Nipro, Japan) reused membranes with a mean QB of 380 B 20 ml/min, QD of 500 ml/min and a mean HD session of 3.5 B 0.5 h. Serum was processed by fluorescence polarization immunoassay for thcy (normal: 10 B 5 Ìmol/l), radioimmunoassay for vitamin B 12 complex including all forms of cobalamins, i.e. Me-Cbl, cyano-cobalamin, hydroxycobalamin and adenosyl-cobalamin (normal: 200 900 pg/ml), and for FA determinations (sfa normal: 110 ; efa normal: 200 700 ). Results are expressed as the mean B SD. Kruskall-Wallis test (one-way ANOVA by ranks) was used to assess differences between groups. Wilcoxon signed ranks test was used to evaluate intragroup differences. p! 0.05 as considered to be significant. Methylcobalamin and Folate Therapy in Hemodialysis Nephron 2002;91:58 63 59

Fig. 1. Percent variations of homocysteine in all groups. Table 2. Initial and final values and percent variations of homocysteine in all groups Group Initial Hcy Ìmol/l Final Hcy Ìmol/l % Hcy variation Group A 22.5B15.6 10.2B3.1 a 44 Group B 19.9B4.0 11.2B1.9 b 43 Group C 25.9B9.3 27.3B9.7 5 Group D 26.6B14.3 24.3B11.8 2.4 Hcy = Homocysteine; Hct = hematocrit. a p = 0.003; b p = 0.012. Results The effects of Me-Cbl and FA on thcy are shown in figure 1 and changes in vitamin levels are summarized in table 3. Effects of Me-Cbl on thcy In group D, final thcy levels (24.3 B 11.8 Ìmol/l) were significantly higher than final levels in groups A (10.2 B 3.1 Ìmol/l, p! 0.001) and B (11.2 B 1.9 Ìmol/l, p! 0.001); no statistical difference with respect to final levels in group C (27.3 B 9.7 Ìmol/l) and with respect to initial values in group D (26.6 B 14.3 Ìmol/l). Effects of FA on thcy Final thcy levels in group B (11.2 B 1.9 Ìmol/l) were similar to final levels of group A (10.2 B 3.1 Ìmol/l) and significantly lower than final levels of groups C (27.3 B 9.7 Ìmol/l, p! 0.001) and D (24.3 B 11.8 Ìmol/l, p! 0.001). Final thcy levels decreased significantly when compared to initial values (11.2 B 1.9 vs. 19.9 B 4.0 Ìmol/l, p = 0.012); percent reduction 43%. Combined Me-Cbl and FA Effects on thcy In group A, thcy final levels were similar to group B (10.2 B 3.1 vs. 11.2 B 1.9 Ìmol/l, p = n.s.) and showed a significant decrease when compared to final levels of groups C (27.3 B 9.7 Ìmol/l, p! 0.001) and D (24.3 B 11.8 Ìmol/l, p! 0.001). Final group A thcy concentrations were significantly lower than initial values (10.2 B 3.1 vs. 22.5 B 15.5 Ìmol/l, p = 0.003); percent decrease 44% (fig. 1). Finally, it was observed that by the first month of treatment (data not shown), 15 patients from group A (90%) but only 11 from group B (70%) had normalized their thcy levels. In the second month, all patients from both groups had normal thcy plasma levels. 60 Nephron 2002;91:58 63 Trimarchi/Schiel/Freixas/Díaz

Table 3. Initial and final levels of vitamin B 12, serum folic acid and erythrocytic folic acid in all groups Group Initial vitamin B 12, pg/ml Final vitamin B 12, pg/ml Initial sfa Final sfa Initial efa Final efa Group A 2,352B1,453 23,553B11,334 a 5.7B2.7 407B422 a 743B847 5,401B1,926 a Group B 2,489B2,423 6,372B5,378 6.6B2.4 267B182 b 485B122 3,259B1,600 b Group C 2,152B1,100 2,205B1,206 7B2.3 6.9B2.2 334B120 316B102 Group D 1,691B1,360 17,422B4,819 c 8.6B3.3 9.7B5.5 778B488 700B439 sfa = Serum folic acid; efa = erythrocytic folic acid. a p = 0.003; b p = 0.012; c p = 0.003. Changes in Vitamin Levels With respect to vitamin levels (table 3), all patients had initial vitamin B 12 values within normal limits and initial low sfa blood levels, despite efa were normal. When Me-Cbl was prescribed, final levels in groups A and D were significantly higher than the initial ones (23,553 B 11,334 vs. 2,352 B 1,453 pmol/ml, p = 0.003 in group A; 17,422 B 4,819 vs. 1,691 B 1,360 pmol/ml, p = 0.003 in group D). Interestingly, despite baseline normal vitamin B 12 and efa blood levels, when considerable high concentrations of Me-Cbl, sfa and efa were achieved after vitamin supplementation, thcy decreased, while thcy actually showed a modest nonsignificant increase in group C. When final values of vitamins were compared between groups, Me-Cbl final levels were significantly higher in group A (p = 0.001) and sfa final levels were significantly higher in groups A and B than in the rest (p = 0.001). Noteworthy, efa levels were significantly higher when Me-Cbl and FA were given together (5,401 B 1,926 ng/ ml, p = 0.001); in group B efa final concentrations were also different (3,258 B 1,600, p = 0.001) from those in groups C (316 B 101 ) and D (699 B 439 ). No adverse effects could be ascribed to Me-Cbl or FA therapy during the study. Discussion Our data show that oral administration of 10 mg FA is safe and highly effective in normalizing thcy blood levels in chronic HD patients, and i.v. Me-Cbl alone did not significantly reduce thcy levels; when prescribed with oral FA supplementation, Me-Cbl achieved a similar decrease in thcy levels than the one obtained with FA alone. Our results emphasize the importance of FA in the regulation of plasma thcy levels in chronic HD patients. A daily oral intake of 5 10 mg FA decreases thcy levels by 20 30%, provided vitamin B 12 levels are within normal limits. A primary function of FA is to participate as a methyl donor for the conversion of homocysteine to methionine. The methyl group is first transferred from methyl-tetrahydrofolate to cobalamin to form Me-Cbl, which then donates the methyl group onto homocysteine to generate methionine [16]. Due to the methylation abnormalities present in chronic renal failure, Me-Cbl concentration may be diminished, and the activity of methionine synthase could be abnormal. This enzyme impairment could result in abnormal DNA synthesis and methylation abnormalities, thereby altering erythropoiesis and protein synthesis, which would then translate into anemia and hypoalbuminemia, respectively. It is also important to emphasize that FA distribution in blood is mainly divided into two compartments: efa, which represents the circulating deposits of FA and was within normal limits in all patients in this study (table 3); and, sfa, the metabolically available or physiologically active compartment, which was low in all groups (table 3), regardless of the time the patients were on HD. After oral FA loading, sfa dramatically increased in all groups and thcy levels normalized (tables 2, 3). Thus, sfa and not efa must be monitored in chronic HD patients to assess its ability to lower thcy levels. With respect to the sole administration of Me-Cbl, albeit vitamin B 12 is important as a cofactor in Hcy metabolism, we were unable to demonstrate its capability to significantly diminish thcy levels when it was prescribed either alone or in combination with FA, despite the remarkable high levels of vitamin B 12 achieved. This may be due to the fact that both cofactors must be present for a correct performance of methionine synthase [16]. Moreover, all HD patients in this study had normal baseline vitamin B 12 concentrations. In one report, the preva- Methylcobalamin and Folate Therapy in Hemodialysis Nephron 2002;91:58 63 61

lence of suboptimal levels of vitamin B 12 in apparently healthy men with HtHcy was approximately 57% [24]. This difference could obviously be because patient characteristics are different but also because total body stores of vitamin B 12 are considerable (approximately 2 5 mg, one-half of which is in the liver). This suggests that from 3 to 5 years must elapse until cobalamin deficiency exists [25] (time on HD of our patient population ranged between 5 months and 6.3 years, average 18.7 months), unless an alteration in vitamin B 12 absorption or prolonged inadequate intake are present [25]. It is probable that in the uncommon situation of vitamin B 12 deficiency, FA alone may not be capable of lowering thcy concentrations either. In addition, the method by which vitamin B 12 blood levels are quantitated is a radioimmunoassay that measures all cobalamins indistinctly, and as methylation abnormalities are present in chronic renal failure [18], Me-Cbl plasma levels could be certainly low, while the total measured amount of cobalamin is normal. Normally, human plasma contains fractions of about 0.6 Me- Cbl, 0.3 adenosyl-cobalamin, and less than 0.1 of hydroxy- and cyano-cobalamin [26]; hydroxy-cobalamin, another cobalamin, is also methylated to form Me-Cbl in human tissue [16]. In relation to this apparent FA-dependent response to lowering thcy levels observed in this study, an intriguing possibility to be analyzed is that thcy may not be a cause of cardiovascular disease or of endothelial dysfunction but otherwise a marker of another risk factor involved in thcy metabolism, as low sfa levels or a decreased physiological bioavailability of FA due to the uremic state. Therefore, the prescription of FA would not only reduce thcy levels (the marker of endothelial dysfunction) but more importantly, provide the FA (if low, the cause of cardiovascular disease) necessary to contribute in the correction of some of the endothelial derangements present in HtHcy. Approximately 70 80% of circulating thcy is bound to large proteins as albumin [4, 27], which is considered as an independent determinant of thcy, unrelated to B vitamin status [1, 27]. However, in our study albumin and hematocrits differences were non-significant among groups (table 1) when simultaneously high vitamin B 12 blood levels were measured, and consequently cannot explain the Me-Cbl failure to correct thcy by itself. In other words, thcy was corrected only when FA was prescribed, either alone (group B) or with Me-Cbl (group D). In addition to vitamin influence, thcy concentration also depends on nutritional intake, genetic factors, on the degree of kidney failure and cellular metabolism and to impairments in the remethylation of homocysteine to methionine [20, 28, 29]. If, for instance, the remethylation reaction is impaired due to vitamin deficiencies or to alterations in the methylation process itself, homocysteine intracellular levels will increase and after its cellular extrusion HtHcy ensues [20]. High intracellular concentrations of homocysteine result in the cellular accumulation of S-adenosyl homocysteine, an extremely toxic compound that inhibits methyltransferases [30]. Several theories suggest that homocysteine causes vascular intimal thickening, elastic lamina disruption, luminal platelet accumulation and increases the proliferation of vascular smooth muscle cells, and hallmarks of atherosclerosis [12, 28, 31]. In contrast with other studies which show that high doses of FA do not normalize Hcy in HD patients [21, 22, 32], our findings demonstrate that with 10 mg FA a day Hcy levels normalize. In patients with chronic renal failure the proposed dose of FA varies between 1 and 15 mg/ day, while vitamin B 12 dose oscilates between 400 Ìg to 1 mg/day [1, 6, 33, 34]. As in other studies, in which Me- Cbl was used to evaluate its effects on diabetic or uremic neuropathy, such high levels were not related to adverse reactions [35, 36]. In addition, some patients have referred improvement of chronic paresthesias. As to the time it takes to normalize thcy plasma levels, it can range from two weeks up to seven months [37 39], depending on the vitamins used, on the doses prescribed and on the population under consideration. Conclusion We believe 10 mg FA can safely be given to chronic HD patients to lower thcy levels, regardless of normal baseline efa plasma levels (sfa may be low). In the setting of normal baseline vitamin B 12 blood levels, the prescription of i.v. Me-Cbl showed no additional benefits to diminish thcy levels. Our study includes a small number of patients; in the absence of data from randomized, controlled trials demonstrating a reduction in cardiovascular outcomes or in thrombotic events with successful treatment of HtHcy in the HD population, careful conclusions must be drawn from our report and the previously published uncontrolled, open-label investigations. Acknowledgements We thank trb pharma s.a. for providing methylcobalamin vials for all patients and Mr. Gabriel Converso for his statistical analysis. 62 Nephron 2002;91:58 63 Trimarchi/Schiel/Freixas/Díaz

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